In recent years polynucleotides have been studied as possible therapeutic agents due to their ability to alter expression of specific genes. Gene therapy to add a gene function which is missing or absent and to inhibit expression of a gene are under investigation. In particular, gene therapy with antisense oligonucleotides is being widely studied. Oligonucleotides are composed of a string of nucleotide residues complementary to the mRNA of a target gene for hybridizing by Watson-Crick base pairing. In this way, inhibition of translation is achieved, often by mechanisms such as activation of RNAse H or by prevention of the assembly or the progress of the translational machinery (Branch, A. D., Hepatology 24(6):1517-1529 (1996)).
One problem with the use of polynucleotides, DNA, RNA and oligonucleotides, as therapeutic agents is a relatively poor ability to cross the cell membrane in order to reach their site of action in the cytoplasm. Polynucleotides carry a negative charge and, therefore, do not readily cross the cell membrane in free form.
Another problem is that polynucleotides can interact with a variety of extracellular molecules which can alter the polynucleotide's bioavailability. Further, polynucleotides are susceptible to degradation in biological fluids and they display pharmacokinetics which may not be favorable for some therapeutic applications.
One approach to overcoming these problems is to administer the polynucleotide in the presence of a lipid vesicle, such as a liposome, and various liposome-based compositions have been proposed (Zelphati, O., and Szoka, F. C., J. Control. Res. 41:99-119 (1996)). For example, one proposed composition consists of a polynucleotide mixed with pre-formed cationic liposomes. Such liposomes are generally prepared from a cationic lipid mixed with an approximately equimolar concentration of a membrane destabilizing lipid and/or a neutral lipid, such as dioleoylphosphatidylethanolamine (DOPE). The polynucleotide is mixed with the pre-formed cationic liposomes to form polynucleotide-liposome complexes through electrostatic charge interactions. Such complexes have some in vitro ability to mediate cellular uptake of polynucleotides, however, the relatively large size (200-2000 nm) and poor stability make them unsuitable for in vivo applications, in particular for delivery to organs other than the liver and the lungs (Bennet, C. F., et al., J. Control. Rel. 41:121-130 (1996); Litzinger, D. C., et al., Biochim. Biophys. Acta 1281:139-149 (1996)).
Another proposed polynucleotide-liposome composition includes a polynucleotide entrapped in the aqueous interior of neutral liposomes formed from neutral vesicle-forming lipids. These liposomes are typically prepared by either hydrating a dried lipid film with a highly concentrated solution of the polynucleotide (Juliano, R. L., and Akhtar, S., Antisense Res. Dev. 2:165-176 (1992)) or by the reverse evaporation method (REV) (Ropert, C., et al., Pharm. Res. 10(10):1427-1433 (1993); Szoka, F. C., and Papahadjopoulos, D., Proc. Natl. Acad. Sci. USA 75(9):4194-4198 (1978)). A problem with polynucleotide-liposome compositions prepared by these methods is a low trapping efficiency of the polynucleotide, in particular for small unilamellar vesicles (&lt;100 nm), where trapping efficiencies on the order of 2-4% have been reported (Zelphati, O., et al., Antiviral Res. 25:13-25 (1994)). Because polynucleotides are expensive, such low trapping efficiencies are unacceptable. The problem is compounded in that recovery of unentrapped polynucleotide can be costly and/or time consuming.